title: Structural perturbation of human hemoglobin on glutathionylation probed by hydrogen-deuterium exchange and MALDI mass spectrometry.
abstract: Glutathionyl hemoglobin, an example of post-translationally modified hemoglobin, has been studied as a marker of oxidative stress in various diseased conditions. Compared to normal hemoglobin, glutathionyl hemoglobin has been found to have increased oxygen affinity and reduced cooperativity. However, detailed information concerning the structural perturbation of hemoglobin associated with glutathionylation is lacking. In the present study, we report structural changes associated with glutathionylation of deoxyhemoglobin by hydrogen/deuterium (H/D) exchange coupled to matrix assisted laser desorption ionization (MALDI) mass spectrometry. We analyzed isotope exchange kinetics of backbone amide hydrogen of eleven peptic peptides in the deoxy state of both hemoglobin and glutathionyl hemoglobin molecules. Analysis of the deuterium incorporation kinetics for both molecules showed structural changes associated with the following peptides: α34-46, α1-29, β32-41, β86-102, β115-129, and β130-146. H/D exchange experiments suggest that glutathionylation of hemoglobin results in a change in conformation located at the above-mentioned regions of the hemoglobin molecule. MALDI mass spectrometry based H/D exchange experiment might be a simple way of monitoring structural changes associated with post-translational modification of protein.
authors.affiliation: Molecular Medicine & Clinical Proteomics, St. John's Research Institute, St. John's National Academy of Health Sciences , Bangalore, India.
authors.forename: Gopa
authors.initials: G
authors.lastname: Mitra
authors.forename: Monita
authors.initials: M
authors.lastname: Muralidharan
authors.forename: Jennifer
authors.initials: J
authors.lastname: Pinto
authors.forename: Krishnamachari
authors.initials: K
authors.lastname: Srinivasan
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Mandal
mesh_terms: D041961:Deuterium Exchange Measurement / Q000379:methods*; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D010100:Oxygen / Q000737:chemistry; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006454:Hemoglobins; C100003:deoxyhemoglobin; D010100:Oxygen